From an organic chemistry standpoint, the process of drug design can be considered to involve two steps. First, a lead chemical template (often one or more) is selected. Second, a synthetic chemistry effort is undertaken to create analogs of the lead chemical template to create a compound or compounds possessing the desired therapeutic and pharmacokinetic properties.
An important step in the drug discovery process is the selection of a suitable lead chemical template upon which to base a chemistry analog program. The process of identifying a lead chemical template for a given molecular target typically involves screening a large number of compounds (often more than 100,000) in a functional assay, selecting a subset based on some arbitrary activity threshold for testing in a secondary assay to confirm activity, and then assessing the remaining active compounds for suitability of chemical elaboration.
This process can be quite time- and resource-consuming, and has numerous disadvantages. It requires the development and implementation of a high-throughput functional assay, which by definition requires that the function of the molecular target be known. It requires the testing of large numbers of compounds, the vast majority of which will be inactive for a given molecular target. It leads to the depletion of chemical resources and requires the continual maintenance of large collections of compounds. Importantly, it often leads to a final pool of potential lead templates that for the most part, with the exception of affinity for a given molecular target, do not possess desirable drug-like qualities. In some cases, high-throughput functional assays do not identify any compounds from the large number (e.g., 100,000) of compounds screened that meet the criteria established for activity.
Thus, what is needed is a faster and better approach to identifying a lead chemical template.
The present invention is related to rational drug design. Specifically, the present invention provides an approach to the development of a library of compounds as well as methods for identifying compounds (e.g., ligands) that bind to a specific target molecule (e.g., proteins) and lead chemical templates that can be used, for example, in drug discovery and design. Significantly and preferably, this approach for identifying ligands for target molecules (e.g., proteins) uses nuclear magnetic resonance (NMR) spectroscopy. There are numerous NMR spectroscopic techniques currently available that detect binding of small molecules to targets such as protein targets, including targets identified using genomics techniques that lack a functional assay. Ligands with only moderate binding affinities, which might be overlooked in a traditional functional assay but yet might serve as templates for subsequent synthetic chemistry efforts, can potentially be identified using the present invention. Preferably, one method of the present invention involves the use of flow NMR techniques, which can reduce the amount of time and effort required to evaluate small molecules for binding to a given target.
In one aspect, the present invention provides a method of creating a chemical compound library, and the library itself. The method includes: selecting compounds having a molecular weight of no greater than about 350 grams/mole; and selecting compounds having a solubility in deuterated water of at least about 1 mM at room temperature. Preferably, a majority (i.e., greater than 50%) of the compounds in the chemical compound library have a molecular weight of no greater than about 350 grams/mole and a solubility in deuterated water of at least about 1 mM at room temperature. More preferably, at least about 75% of the compounds, and most preferably, all of the compounds in the chemical compound library have a molecular weight of no greater than about 350 grams/mole and a solubility in deuterated water of at least about 1 mM at room temperature. Preferably, this library of compounds includes at least about 75 compounds, more preferably, at least about 300 compounds, and most preferably, at least about 2000 compounds, and have relatively diverse chemical structures. Herein, the molecular weights of the compounds are determined without solubilizing counterions (if the compounds are salts) and without water molecules of hydration. Also, concentrations are reported based on aqueous solutions, which may or may not include a buffer.
In another embodiment, the present invention provides a method of identifying a lead chemical template (of which there often may be one or more), for example, for designing a bioactive agent such as a drug (e.g., a compound having therapeutic and/or prophylactic capabilities). The method includes: selecting compounds having a molecular weight of no greater than about 350 grams/mole, and a solubility in deuterated water of at least about 1 mM at room temperature to create a chemical compound library; identifying at least one compound from the library that functions as a ligand (i.e., a compound that binds to a target molecule) having a dissociation constant to a target molecule (e.g., protein) of no weaker than (i.e., at least) about 100 xcexcM; and using the ligand to identify a lead chemical template, which can be used, for example, for designing a drug. Preferably, the lead chemical template has a dissociation constant to a target molecule (e.g., protein) of no weaker than (i.e., at least) about 1 xcexcM. Preferably, the lead chemical template can be identified through further screening efforts or through direct chemical elaborations. Preferably, a majority (i.e., greater than 50%) of the compounds in the chemical compound library, more preferably, at least about 75%, and most preferably, all of the compounds in the chemical compound library, have a molecular weight of no greater than about 350 grams/mole and a solubility in deuterated water of at least about 1 mM at room temperature.
Another embodiment of the present invention provides a method of identifying a compound that binds to a target molecule (e.g., protein). The method includes: providing a plurality of mixtures of test compounds, each mixture being in a (separate) sample reservoir (preferably, a sample reservoir of a multiwell sample holder (e.g., a 96-well microtiter plate)); introducing a target molecule (e.g., protein) into each of the sample reservoirs to provide a plurality of test samples; providing a nuclear magnetic spectrometer equipped with a flow-injection probe; transferring each test sample from the sample reservoir into the flow-injection probe; collecting a relaxation-edited (preferably, a one-dimensional (1D) relaxation-edited) nuclear magnetic resonance spectrum (preferably, a 1H NMR spectrum) on each sample in each reservoir; and comparing the spectra of each sample to the spectra taken under the same conditions in the absence of the target molecule (e.g., protein) to identify compounds that bind to the target molecule (e.g., protein); wherein the concentration of target molecule (e.g., protein) and each compound in each sample is no greater than about 100 xcexcM. Preferably, the mixture of compounds comprises at least about 3 compounds (more preferably, at least about 6 compounds, and most preferably, at least about 10 compounds), each having at least one distinguishable resonance in an NMR spectrum (preferably, a 1D NMR spectrum, and more preferably, a 1D 1H NMR spectrum) of the mixture.
Preferably, in this method, the ratio of target molecule (e.g., protein) to compounds in each sample reservoir is about 1:1. More preferably, the concentration of target molecule (e.g., protein) and each compound in each sample is at least about 25 xcexcM. Most preferably, the concentration of target molecule (e.g., protein) and each compound in each sample is no greater than about 50 xcexcM.
Sample requirements can be reduced even further if WaterLOGSY (water-ligand observation with gradient spectroscopy) methods are used as an alternative to the relaxation-editing method described above to detect the binding interaction.
The present invention provides yet another method of identifying a compound that binds to a target molecule (e.g., protein). This method includes: providing a plurality of mixtures of test compounds, each mixture being in a sample reservoir; introducing a target molecule into each of the sample reservoirs to provide a plurality of test samples; providing a nuclear magnetic resonance spectrometer equipped with a flow-injection probe; transferring each test sample from the sample reservoir into the flow-injection probe; collecting a WaterLOGSY nuclear magnetic resonance spectrum (preferably, a 1D WaterLOGSY nuclear magnetic resonance spectrum) on each sample in each reservoir; and analyzing the spectra of each sample to distinguish binding compounds from nonbinding compounds by virtue of the opposite sign of their water-ligand nuclear Overhauser effects (NOEs). Preferably, the concentration of each compound in each sample is no greater than about 100 xcexcM, although higher concentrations can be used if desired.
In this method when binding is detected using the WaterLOGSY technique, extremely low levels of target can be used with ratios of ligand to target of about 100:1 to about 10:1. Preferably, the concentration of target molecule is no greater than about 10 xcexcM. More preferably, the concentration of target molecule is about 1 xcexcM to about 10 xcexcM. For data analysis, binding compounds are distinguished from nonbinders (i.e., nonbinding compounds) by the opposite sign of their water-ligand NOEs. With this method, there is no need to collect a reference spectrum in the absence of a target molecule.
In preferred embodiments of the present invention, a majority of the compounds in the library have a solubility in deuterated water of at least about 1 mM at room temperature (i.e., about 25xc2x0 C. to about 30xc2x0 C.), and a molecular weight of no greater than about 350 grams/mole. For effective use of a compound identified as a ligand for a given target in the search for a lead chemical template, preferably, the dissociation constant of the identified ligand to a target molecule is no weaker than (i.e., at least) about 100 xcexcM. For effective use of a lead chemical template in further drug design, preferably, the dissociation constant for the lead chemical template to a target molecule is no weaker than (i.e., at least) about 1 xcexcM.
In another aspect, the invention provides a method of identifying a protein function. The method includes providing a plurality of mixtures of test compounds, each mixture being in a sample reservoir and containing a plurality of test compounds; introducing a target molecule into each of the sample reservoirs to provide a plurality of test samples; providing a nuclear magnetic resonance spectrometer equipped with a flow-injection probe; transferring each test sample from the sample reservoir into the flow-injection probe; collecting a relaxation-edited nuclear magnetic resonance spectrum on each sample in each reservoir; comparing the spectra of each sample to the spectra taken under the same conditions in the absence of the target molecule to identify compounds that bind to the target molecule, wherein the concentration of target molecule and each compound in each sample is no greater than about 100 xcexcM; and determining a function of the target molecule based upon known binding characteristics of the test compounds that bind to the target molecule.